Diversity-generating retro elements (DGRs) were discovered in Bordetella bacteriophage on the basis of their ability to generate vast amounts of diversity in target genes. They function through a template-dependent, reverse transcriptase-mediated mechanism that introduces nucleotide substitutions at specified sites in protein-coding sequences. Variable residues are displayed in the ligand-binding pocket of a specialized scaffold which balances protein diversity with structural stability, creating vast repertoires of receptors for potential ligand-receptor interactions. Using the Bordetella phage DGR as a template, we have identified homologous elements in numerous bacterial, plasmid, and bacteriophage genomes. Most DGRs are bacterial chromosomal elements and they are distributed throughout the bacterial domain. Of particular relevance to health and disease, DGRs are present as chromosomal elements in human commensals including members of the Bacteroides, Bifid bacterium, Eubacterium and Ruminococcus genera, and they are also encoded by pathogens such as Legionella pneumophila, Treponema denticola, and Bacteroides fragilis. Despite their widespread distribution in nature, our understanding of the mechanisms of DGR function and the selective advantages they confer is at a rudimentary stage. This application focuses on two DGR systems which provide complementary components of an experimental platform that will allow us to address some of the most compelling unanswered questions regarding these elements. The Bordetella BPP-1 phage DGR is by far the best characterized and it provides a paradigm for all members of this retro element family. L. pneumophila is a well studied pathogen amenable to genetic manipulation and it has become our prototype system for studying bacterial DGRs. Our specific aims are as follows: 1. Determine the mechanisms of adenine mutagenesis and retro homing by the Bordetella phage DGR. These are the hallmarks of DGR activity and they are essential for the creation of diversity. 2. Probe variable protein localization and structure, and the regulation of diversity by a L. pneumophila DGR. We will test the hypothesis that DGRs have been exploited for bacterial surface display of variable protein repertoires. Understanding DGRs will reveal new mechanisms for accelerated evolution by bacteria and phage and provide new paradigms for understanding adaptations of importance to human health and disease. PUBLIC HEALTH RELEVANCE: Our studies will contribute to an understanding of a newly discovered mechanism that is widely used by bacteria and bacteriophage to generate massive amounts of protein diversity to accelerate the evolution of adaptive traits. Our proposed experiments involve important pathogens belonging to the Bordetella and Legionella genera.